The present invention relates generally to the fields of organic chemistry and biology. In particular, the present invention is directed to compositions and methods for use in oligonucleotide synthesis.
Phosphoramidite chemistry [Beaucage, S. L. and lyer, R. P. Tetrahedron 48, 2223-2311 (1992)] has become by far the most widely used coupling chemistry for the synthesis of oligonucleotides. As is well known to those skilled in the art, phosphoramidite synthesis of oligonucleotides involves activation of nucleoside phosphoramidite monomer precursors by reaction with an activating agent to form activated intermediates, followed by sequential addition of the activated intermediates to the growing oligonucleotide chain (generally anchored at one end to a suitable solid support) to form the oligonucleotide product. Tetrazole is commonly used for the activation of the nucleoside phosphoramidite monomers; the activation occurs by the mechanism depicted in Scheme I. Tetrazole has an acidic proton which presumably protonates the basic nitrogen of the diisopropylamino phosphine group, thus making the diisopropylamino group a leaving group. The negatively charged tetrazolium ion then makes an attack on the trivalent phosphorous, forming a transient phosphorous tetrazolide species. The 5'--OH group of the solid support bound nucleoside then attacks the active trivalent phosphorous species, resulting in the formation of the internucleotide linkage. The trivalent phosphorous is finally oxidized to the pentavalent phosphorous. ##STR1##
A principal drawback of tetrazole is its cost. It is the second most expensive reagent in oligonucleotide synthesis, costing about 40-50% the price of the nucleoside phosphoramidite. Because of the inherent instability of this highly nitrogenous heterocyclic compound, moreover, sublimed tetrazole is generally required to ensure desired coupling yields. Further, tetrazole (which is typically useo near its saturated solubility of 0.5M) tends to precipitate out of acetonitrile solution at cold temperatures; this can lead to valve blockage on some automated DNA synthesizers.
Other activators which work almost as efficiently as tetrazole have similar drawbacks to those of tetrazole as discussed above. These activators include the following members of the tetrazole class of activators: 5-(p-nitrophenyl) tetrazole [Froehler, B. C. & Matteucci, M. D., Tetrahedron Letters 24, 3171-3174 (1983)]; 5-(p-nitrophenyl) tetrazole+DMAP [Pon, R. T., Tetrahedron Letters 28, 3643-3646 (1987); and 5-(ethylthio)-1-H-tetrazole [Wright, P. et al., Tetrahedron Letters 34, 3373-3376 (1993). In addition to the tetrazole class of activators, the following activators have been employed: N-methylaniline trifluoroacetate [Fourray, J. L. & Varenne, J., Tetrahedron Letters 25, 4511-4514 (1984)]; N-methyl anilinium trichloroacetate [Fourrey, J. L. et al., Tetrahedron Letters 28, 1769-1772 (1987)]; 1-methylimidazoletrifluoromethane sulfonate [Arnold, L. et al., Collect. Czech. Chem. Commun. 54, 523-532 (1989)]; octanoic acid or triethylamine [Stec, W. J. & Zon, G., Tetrahedron Letters 25, 5279-5282 (1984)]; 1-methylimidazole. HCl, 5-trifluoromethyl-1H-tetrazole, N,N-dimethylaniline. HCl and N,N-dimethylaminopyridine. HCl [Hering, G. et al., Nucleosides and Nucleotides 4, 169-171 (1985)]. Overall, these activators gave inferior performance relative to tetrazole.
It is an object of the present invention to provide activated nucleosides for use in solid phase synthesis which do not exhibit all of the drawbacks of the prior art compositions.
It is a further object of the present invention to provide methods for the preparation and use of activated nucleosides as hereinafter described.